Tuberculosis remains a serious health concern. A major virulence property of Mycobacterium tuberculosis, the bacterium responsible for this disease, is the ability to survive and grow within macrophages. M. tuberculosis proteins that are exported out from the cytoplasm to the bacterial surface or are further secreted into the environment are ideally positioned to interact with the host and combat macrophage defenses. Many of the M. tuberculosis proteins in this subcellular exported category remain to be identified or studied. We recently developed a genetic reporter system for monitoring protein export directly in M. tuberculosis. In this system a truncated ?-lactamase enzyme, which is not exported on its own, is fused to proteins to report on their export beyond the cytoplasm. Because ?- lactam antibiotics target cell wall synthesis enzymes, the ?-lactamase reporter must be exported in order to protect bacteria from these drugs. We demonstrated this system to function properly in a ?-lactam sensitive mutant of M. tuberculosis. In this R21 application we propose to use a modified Himar mariner transposon which carries a ?-lactamase reporter, Tn'blaTEM-1, to select for in-frame transposon insertions in M. tuberculosis genes encoding exported proteins. This genetic approach has the advantage of simultaneously identifying exported proteins and generating M. tuberculosis transposon insertion mutants in the corresponding genes. The resulting transposon mutant library will be screened for intracellular growth defects in macrophages and for defects in the M. tuberculosis inhibition of macrophage responses. This research will complement existing genomic approaches while uncovering new virulence factors, which should aid development of new disease intervention strategies. Tuberculosis is a severe world health problem. A virulence property of Mycobacterium tuberculosis, the causative agent of this disease, is the ability to survive and grow in macrophages. The proposed research will identify new virulence factors that enable M. tuberculosis to survive in macrophages and cause disease